De Novo Elastin Assembly Alleviates Development of Supravalvular Aortic Stenosis-Brief Report.

BACKGROUND: A series of incurable cardiovascular disorders arise due to improper formation of elastin during development. Supravalvular aortic stenosis (SVAS), resulting from a haploinsufficiency of ELN, is caused by improper stress sensing by medial vascular smooth muscle cells, leading to progressive luminal occlusion and heart failure. SVAS remains incurable, as current therapies do not address the root issue of defective elastin. METHODS: We use SVAS here as a model of vascular proliferative disease using both human induced pluripotent stem cell-derived vascular smooth muscle cells and developmental Eln+/- mouse models to establish de novo elastin assembly as a new therapeutic intervention. RESULTS: We demonstrate mitigation of vascular proliferative abnormalities following de novo extracellular elastin assembly through the addition of the polyphenol epigallocatechin gallate to SVAS human induced pluripotent stem cell-derived vascular smooth muscle cells and in utero to Eln+/- mice. CONCLUSIONS: We demonstrate de novo elastin deposition normalizes SVAS human induced pluripotent stem cell-derived vascular smooth muscle cell hyperproliferation and rescues hypertension and aortic mechanics in Eln+/- mice, providing critical preclinical findings for the future application of epigallocatechin gallate treatment in humans.

Macrophage-Derived 25-Hydroxycholesterol Promotes Vascular Inflammation, Atherogenesis, and Lesion Remodeling.

BACKGROUND: Cross-talk between sterol metabolism and inflammatory pathways has been demonstrated to significantly affect the development of atherosclerosis. Cholesterol biosynthetic intermediates and derivatives are increasingly recognized as key immune regulators of macrophages in response to innate immune activation and lipid overloading. 25-Hydroxycholesterol (25-HC) is produced as an oxidation product of cholesterol by the enzyme cholesterol 25-hydroxylase (CH25H) and belongs to a family of bioactive cholesterol derivatives produced by cells in response to fluctuating cholesterol levels and immune activation. Despite the major role of 25-HC as a mediator of innate and adaptive immune responses, its contribution during the progression of atherosclerosis remains unclear. METHODS: The levels of 25-HC were analyzed by liquid chromatography-mass spectrometry, and the expression of CH25H in different macrophage populations of human or mouse atherosclerotic plaques, respectively. The effect of CH25H on atherosclerosis progression was analyzed by bone marrow adoptive transfer of cells from wild-type or Ch25h-/- mice to lethally irradiated Ldlr-/- mice, followed by a Western diet feeding for 12 weeks. Lipidomic, transcriptomic analysis and effects on macrophage function and signaling were analyzed in vitro from lipid-loaded macrophage isolated from Ldlr-/- or Ch25h-/-;Ldlr-/- mice. The contribution of secreted 25-HC to fibrous cap formation was analyzed using a smooth muscle cell lineage-tracing mouse model, Myh11ERT2CREmT/mG;Ldlr-/-, adoptively transferred with wild-type or Ch25h-/- mice bone marrow followed by 12 weeks of Western diet feeding. RESULTS: We found that 25-HC accumulated in human coronary atherosclerotic lesions and that macrophage-derived 25-HC accelerated atherosclerosis progression, promoting plaque instability through autocrine and paracrine actions. 25-HC amplified the inflammatory response of lipid-loaded macrophages and inhibited the migration of smooth muscle cells within the plaque. 25-HC intensified inflammatory responses of lipid-laden macrophages by modifying the pool of accessible cholesterol in the plasma membrane, which altered Toll-like receptor 4 signaling, promoted nuclear factor-κB-mediated proinflammatory gene expression, and increased apoptosis susceptibility. These effects were independent of 25-HC-mediated modulation of liver X receptor or SREBP (sterol regulatory element-binding protein) transcriptional activity. CONCLUSIONS: Production of 25-HC by activated macrophages amplifies their inflammatory phenotype, thus promoting atherogenesis.

FN (Fibronectin)-Integrin α5 Signaling Promotes Thoracic Aortic Aneurysm in a Mouse Model of Marfan Syndrome.

BACKGROUND: Marfan syndrome, caused by mutations in the gene for fibrillin-1, leads to thoracic aortic aneurysms (TAAs). Phenotypic modulation of vascular smooth muscle cells (SMCs) and ECM (extracellular matrix) remodeling are characteristic of both nonsyndromic and Marfan aneurysms. The ECM protein FN (fibronectin) is elevated in the tunica media of TAAs and amplifies inflammatory signaling in endothelial and SMCs through its main receptor, integrin α5ß1. We investigated the role of integrin α5-specific signals in Marfan mice in which the cytoplasmic domain of integrin α5 was replaced with that of integrin α2 (denoted α5/2 chimera). METHODS: We crossed α5/2 chimeric mice with Fbn1mgR/mgR mice (mgR model of Marfan syndrome) to evaluate the survival rate and pathogenesis of TAAs among wild-type, α5/2, mgR, and α5/2 mgR mice. Further biochemical and microscopic analysis of porcine and mouse aortic SMCs investigated molecular mechanisms by which FN affects SMCs and subsequent development of TAAs. RESULTS: FN was elevated in the thoracic aortas from Marfan patients, in nonsyndromic aneurysms, and in mgR mice. The α5/2 mutation greatly prolonged survival of Marfan mice, with improved elastic fiber integrity, mechanical properties, SMC density, and SMC contractile gene expression. Furthermore, plating of wild-type SMCs on FN decreased contractile gene expression and activated inflammatory pathways whereas α5/2 SMCs were resistant. These effects correlated with increased NF-kB activation in cultured SMCs and mgR aortas, which was alleviated by the α5/2 mutation or NF-kB inhibition. CONCLUSIONS: FN-integrin α5 signaling is a significant driver of TAA in the mgR mouse model. This pathway thus warrants further investigation as a therapeutic target.

Desmosterol suppresses macrophage inflammasome activation and protects against vascular inflammation and atherosclerosis.

Cholesterol biosynthetic intermediates, such as lanosterol and desmosterol, are emergent immune regulators of macrophages in response to inflammatory stimuli or lipid overloading, respectively. However, the participation of these sterols in regulating macrophage functions in the physiological context of atherosclerosis, an inflammatory disease driven by the accumulation of cholesterol-laden macrophages in the artery wall, has remained elusive. Here, we report that desmosterol, the most abundant cholesterol biosynthetic intermediate in human coronary artery lesions, plays an essential role during atherogenesis, serving as a key molecule integrating cholesterol homeostasis and immune responses in macrophages. Depletion of desmosterol in myeloid cells by overexpression of 3ß-hydroxysterol Δ24-reductase (DHCR24), the enzyme that catalyzes conversion of desmosterol to cholesterol, promotes the progression of atherosclerosis. Single-cell transcriptomics in isolated CD45+CD11b+ cells from atherosclerotic plaques demonstrate that depletion of desmosterol increases interferon responses and attenuates the expression of antiinflammatory macrophage markers. Lipidomic and transcriptomic analysis of in vivo macrophage foam cells demonstrate that desmosterol is a major endogenous liver X receptor (LXR) ligand involved in LXR/retinoid X receptor (RXR) activation and thus macrophage foam cell formation. Decreased desmosterol accumulation in mitochondria promotes macrophage mitochondrial reactive oxygen species production and NLR family pyrin domain containing 3 (NLRP3)-dependent inflammasome activation. Deficiency of NLRP3 or apoptosis-associated speck-like protein containing a CARD (ASC) rescues the increased inflammasome activity and atherogenesis observed in desmosterol-depleted macrophages. Altogether, these findings underscore the critical function of desmosterol in the atherosclerotic plaque to dampen inflammation by integrating with macrophage cholesterol metabolism and inflammatory activation and protecting from disease progression.

Muscle LIM Protein Force-Sensing Mediates Sarcomeric Biomechanical Signaling in Human Familial Hypertrophic Cardiomyopathy.

BACKGROUND: Familial hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease and is typically caused by mutations in genes encoding sarcomeric proteins that regulate cardiac contractility. HCM manifestations include left ventricular hypertrophy and heart failure, arrythmias, and sudden cardiac death. How dysregulated sarcomeric force production is sensed and leads to pathological remodeling remains poorly understood in HCM, thereby inhibiting the efficient development of new therapeutics. METHODS: Our discovery was based on insights from a severe phenotype of an individual with HCM and a second genetic alteration in a sarcomeric mechanosensing protein. We derived cardiomyocytes from patient-specific induced pluripotent stem cells and developed robust engineered heart tissues by seeding induced pluripotent stem cell-derived cardiomyocytes into a laser-cut scaffold possessing native cardiac fiber alignment to study human cardiac mechanobiology at both the cellular and tissue levels. Coupled with computational modeling for muscle contraction and rescue of disease phenotype by gene editing and pharmacological interventions, we have identified a new mechanotransduction pathway in HCM, shown to be essential in modulating the phenotypic expression of HCM in 5 families bearing distinct sarcomeric mutations. RESULTS: Enhanced actomyosin crossbridge formation caused by sarcomeric mutations in cardiac myosin heavy chain (MYH7) led to increased force generation, which, when coupled with slower twitch relaxation, destabilized the MLP (muscle LIM protein) stretch-sensing complex at the Z-disc. Subsequent reduction in the sarcomeric muscle LIM protein level caused disinhibition of calcineurin-nuclear factor of activated T-cells signaling, which promoted cardiac hypertrophy. We demonstrate that the common muscle LIM protein-W4R variant is an important modifier, exacerbating the phenotypic expression of HCM, but alone may not be a disease-causing mutation. By mitigating enhanced actomyosin crossbridge formation through either genetic or pharmacological means, we alleviated stress at the Z-disc, preventing the development of hypertrophy associated with sarcomeric mutations. CONCLUSIONS: Our studies have uncovered a novel biomechanical mechanism through which dysregulated sarcomeric force production is sensed and leads to pathological signaling, remodeling, and hypertrophic responses. Together, these establish the foundation for developing innovative mechanism-based treatments for HCM that stabilize the Z-disc MLP-mechanosensory complex.

Evolving Mural Defects, Dilatation, and Biomechanical Dysfunction in Angiotensin II-Induced Thoracic Aortopathies.

BACKGROUND: Thoracic aortopathy associates with extracellular matrix remodeling and altered biomechanical properties. We sought to quantify the natural history of thoracic aortopathy in a common mouse model and to correlate measures of wall remodeling such as aortic dilatation or localized mural defects with evolving microstructural composition and biomechanical properties of the wall. METHODS: We combined a high-resolution multimodality imaging approach (panoramic digital image correlation and optical coherence tomography) with histopathologic examinations and biaxial mechanical testing to correlate spatially, for the first time, macroscopic mural defects and medial degeneration within the ascending aorta with local changes in aortic wall composition and mechanical properties. RESULTS: Findings revealed strong correlations between local decreases in elastic energy storage and increases in circumferential material stiffness with increasing proximal aortic diameter and especially mural defect size. Mural defects tended to exhibit a pronounced biomechanical dysfunction that is driven by an altered organization of collagen and elastic fibers. CONCLUSIONS: While aneurysmal dilatation is often observed within particular segments of the aorta, dissection and rupture initiate as highly localized mechanical failures. We show that wall composition and material properties are compromised in regions of local mural defects, which further increases the dilatation and overall structural vulnerability of the wall. Identification of therapies focused on promoting robust collagen accumulation may protect the wall from these vulnerabilities and limit the incidence of dissection and rupture.

Endothelial Cell TGF-ß (Transforming Growth Factor-Beta) Signaling Regulates Venous Adaptive Remodeling to Improve Arteriovenous Fistula Patency.

BACKGROUND: Arteriovenous fistulae (AVF) are the gold standard for vascular access for hemodialysis. Although the vein must thicken and dilate for successful hemodialysis, excessive wall thickness leads to stenosis causing AVF failure. Since TGF-ß (transforming growth factor-beta) regulates ECM (extracellular matrix) deposition and smooth muscle cell (SMC) proliferation-critical components of wall thickness-we hypothesized that disruption of TGF-ß signaling prevents excessive wall thickening during venous remodeling. METHODS: A mouse aortocaval fistula model was used. SB431542-an inhibitor of TGF-ß receptor I-was encapsulated in nanoparticles and applied to the AVF adventitia in C57BL/6J mice. Alternatively, AVFs were created in mice with conditional disruption of TGF-ß receptors in either SMCs or endothelial cells. Doppler ultrasound was performed serially to confirm patency and to measure vessel diameters. AVFs were harvested at predetermined time points for histological and immunofluorescence analyses. RESULTS: Inhibition of TGF-ß signaling with SB431542-containing nanoparticles significantly reduced p-Smad2-positive cells in the AVF wall during the early maturation phase (days 7-21) and was associated with decreased AVF wall thickness that showed both decreased collagen density and decreased SMC proliferation. SMC-specific TGF-ß signaling disruption decreased collagen density but not SMC proliferation or wall thickness. Endothelial cell-specific TGF-ß signaling disruption decreased both collagen density and SMC proliferation in the AVF wall and was associated with reduced wall thickness, increased outward remodeling, and improved AVF patency. CONCLUSIONS: Endothelial cell-targeted TGF-ß inhibition may be a translational strategy to improve AVF patency.

The role of LTBPs in TGF beta signaling.

The purpose of this review is to discuss the transforming growth factor beta (TGFB) binding proteins (LTBP) with respect to their participation in the activity of TGFB. We first describe pertinent aspects of the biology and cell function of the LTBPs. We then summarize the physiological consequences of LTBP loss in humans and mice. Finally, we consider a number of outstanding questions relating to LTBP function.

Roles of mTOR in thoracic aortopathy understood by complex intracellular signaling interactions.

Thoracic aortopathy-aneurysm, dissection, and rupture-is increasingly responsible for significant morbidity and mortality. Advances in medical genetics and imaging have improved diagnosis and thus enabled earlier prophylactic surgical intervention in many cases. There remains a pressing need, however, to understand better the underlying molecular and cellular mechanisms with the hope of finding robust pharmacotherapies. Diverse studies in patients and mouse models of aortopathy have revealed critical changes in multiple smooth muscle cell signaling pathways that associate with disease, yet integrating information across studies and models has remained challenging. We present a new quantitative network model that includes many of the key smooth muscle cell signaling pathways and validate the model using a detailed data set that focuses on hyperactivation of the mechanistic target of rapamycin (mTOR) pathway and its inhibition using rapamycin. We show that the model can be parameterized to capture the primary experimental findings both qualitatively and quantitatively. We further show that simulating a population of cells by varying receptor reaction weights leads to distinct proteomic clusters within the population, and that these clusters emerge due to a bistable switch driven by positive feedback in the PI3K/AKT/mTOR signaling pathway.

Developmental origins of mechanical homeostasis in the aorta.

BACKGROUND: Mechanical homeostasis promotes proper aortic structure and function. Pathological conditions may arise, in part, from compromised or lost homeostasis. There is thus a need to quantify the homeostatic state and when it emerges. Here we quantify changes in mechanical loading, geometry, structure, and function of the murine aorta from the late prenatal period into maturity. RESULTS: Our data suggest that a homeostatic set-point is established by postnatal day P2 for the flow-induced shear stress experienced by endothelial cells; this value deviates from its set-point from P10 to P21 due to asynchronous changes in mechanical loading (flow, pressure) and geometry (radius, wall thickness), but is restored thereafter consistent with homeostasis. Smooth muscle contractility also decreases during this period of heightened matrix deposition but is also restored in maturity. The pressure-induced mechanical stress experienced by intramural cells initially remains low despite increasing blood pressure, and then increases while extracellular matrix accumulates. CONCLUSIONS: These findings suggest that cell-level mechanical homeostasis emerges soon after birth to allow mechanosensitive cells to guide aortic development, with deposition of matrix after P2 increasingly stress shielding intramural cells. The associated tissue-level set-points that emerge for intramural stress can be used to assess and model the aorta that matures biomechanically by P56.

Endothelial Cell-Derived Interleukin-18 Released During Ischemia Reperfusion Injury Selectively Expands T Peripheral Helper Cells to Promote Alloantibody Production.

BACKGROUND: Ischemia reperfusion injury (IRI) predisposes to the formation of donor-specific antibodies, a factor contributing to chronic rejection and late allograft loss. METHODS: We describe a mechanism underlying the correlative association between IRI and donor-specific antibodies by using humanized models and patient specimens. RESULTS: IRI induces immunoglobulin M-dependent complement activation on endothelial cells that assembles an NLRP3 (NOD-like receptor pyrin domain-containing protein 3) inflammasome via a Rab5-ZFYVE21-NIK axis and upregulates ICOS-L (inducible costimulator ligand) and PD-L2 (programmed death ligand 2). Endothelial cell-derived interleukin-18 (IL-18) selectively expands a T-cell population (CD4+CD45RO+PD-1hiICOS+CCR2+CXCR5-) displaying features of recently described T peripheral helper cells. This population highly expressed IL-18R1 and promoted donor-specific antibodies in response to IL-18 in vivo. In patients with delayed graft function, a clinical manifestation of IRI, these cells were Ki-67+IL-18R1+ and could be expanded ex vivo in response to IL-18. CONCLUSIONS: IRI promotes elaboration of IL-18 from endothelial cells to selectively expand alloreactive IL-18R1+ T peripheral helper cells in allograft tissues to promote donor-specific antibody formation.

H19/TET1 axis promotes TGF-ß signaling linked to endothelial-to-mesenchymal transition.

While emerging evidence suggests the link between endothelial activation of TGF-ß signaling, induction of endothelial-to-mesenchymal transition (EndMT), and cardiovascular disease (CVD), the molecular underpinning of this connection remains enigmatic. Here, we report aberrant expression of H19 lncRNA and TET1 in endothelial cells (ECs) of human atherosclerotic coronary arteries. Using primary human umbilical vein endothelial cells (HUVECs) and aortic endothelial cells (HAoECs) we show that TNF-α, a known risk factor for endothelial dysfunction and CVD, induces H19 expression which in turn activates TGF-ß signaling and EndMT via a TET1-dependent epigenetic mechanism. We also show that H19 regulates TET1 expression at the posttranscriptional level. Further, we provide evidence that this H19/TET1-mediated regulation of TGF-ß signaling and EndMT occurs in mouse pulmonary microvascular ECs in vivo under hyperglycemic conditions. We propose that endothelial activation of the H19/TET1 axis may play an important role in EndMT and perhaps CVD.

Complement Membrane Attack Complexes Assemble NLRP3 Inflammasomes Triggering IL-1 Activation of IFN-γ-Primed Human Endothelium.

RATIONALE: Complement activation contributes to multiple immune-mediated pathologies. In late allograft failure, donor-specific antibody deposits complement membrane attack complexes (MAC) on graft endothelial cells (ECs), substantially increasing their immunogenicity without causing lysis. Internalized MAC stabilize NIK (NF-κB [nuclear factor kappa-light-chain-enhancer of activated B cells]-inducing kinase) protein on Rab5+MAC+ endosomes, activating noncanonical NF-κB signaling. However, the link to increased immunogenicity is unclear. OBJECTIVE: To identify mechanisms by which alloantibody and internalized MAC activate ECs to enhance their ability to increase T-cell responses. METHODS AND RESULTS: In human EC cultures, internalized MAC also causes NLRP3 (NOD-like receptor family pyrin domain containing 3) translocation from endoplasmic reticulum to Rab5+MAC+NIK+ endosomes followed by endosomal NIK-dependent inflammasome assembly. Cytosolic NIK, stabilized by LIGHT (lymphotoxin-like inducible protein that competes with glycoprotein D for herpesvirus entry on T cells), does not trigger inflammasome assembly, and ATP-triggered inflammasome assembly does not require NIK. IFN-γ (interferon-γ) primes EC responsiveness to MAC by increasing NLRP3, pro-caspase 1, and gasdermin D expression. NIK-activated noncanonical NF-κB signaling induces pro-IL (interleukin)-1ß expression. Inflammasome processed pro-IL-1ß, and gasdermin D results in IL-1ß secretion that increases EC immunogenicity through IL-1 receptor signaling. Activation of human ECs lining human coronary artery grafts in immunodeficient mouse hosts by alloantibody and complement similarly depends on assembly of an NLRP3 inflammasome. Finally, in renal allograft biopsies showing chronic rejection, caspase-1 is activated in C4d+ ECs of interstitial microvessels, supporting the relevance of the cell culture findings. CONCLUSIONS: In response to antibody-mediated complement activation, IFN-γ-primed human ECs internalize MAC, triggering both endosomal-associated NIK-dependent NLRP3 inflammasome assembly and IL-1 synthesis, resulting in autocrine/paracrine IL-1ß-mediated increases in EC immunogenicity. Similar responses may underlie other complement-mediated pathologies.

Caveolin-1 Regulates Atherogenesis by Attenuating Low-Density Lipoprotein Transcytosis and Vascular Inflammation Independently of Endothelial Nitric Oxide Synthase Activation.

BACKGROUND: Atherosclerosis is driven by synergistic interactions between pathological, biomechanical, inflammatory, and lipid metabolic factors. Our previous studies demonstrated that absence of caveolin-1 (Cav1)/caveolae in hyperlipidemic mice strongly inhibits atherosclerosis, which was attributed to activation of endothelial nitric oxide (NO) synthase (eNOS) and increased production of NO and reduced inflammation and low-density lipoprotein trafficking. However, the contribution of eNOS activation and NO production in the athero-protection of Cav1 and the exact mechanisms by which Cav1/caveolae control the pathogenesis of diet-induced atherosclerosis are still not clear. METHODS: Triple-knockout mouse lacking expression of eNOS, Cav1, and Ldlr were generated to explore the role of NO production in Cav1-dependent athero-protective function. The effects of Cav1 on lipid trafficking, extracellular matrix remodeling, and vascular inflammation were studied both in vitro and in vivo with a mouse model of diet-induced atherosclerosis. The expression of Cav1 and distribution of caveolae regulated by flow were analyzed by immunofluorescence staining and transmission electron microscopy. RESULTS: We found that absence of Cav1 significantly suppressed atherogenesis in Ldlr-/-eNOS-/- mice, demonstrating that athero-suppression is independent of increased NO production. Instead, we find that the absence of Cav1/caveolae inhibited low-density lipoprotein transport across the endothelium and proatherogenic fibronectin deposition and disturbed flow-mediated endothelial cell inflammation. Consistent with the idea that Cav1/caveolae may play a role in early flow-dependent inflammatory priming, distinct patterns of Cav1 expression and caveolae distribution were observed in athero-prone and athero-resistant areas of the aortic arch even in wild-type mice. CONCLUSIONS: These findings support a role for Cav1/caveolae as a central regulator of atherosclerosis that links biomechanical, metabolic, and inflammatory pathways independently of endothelial eNOS activation and NO production.

ABO blood group does not impact incidence or outcomes of surgery for acute type A aortic dissection.

Objectives. To evaluate the distribution and impact of ABO blood groups on postoperative outcomes in patients undergoing surgery for acute type A aortic dissection (ATAAD). Design. A total of 1144 surgical ATAAD patients from eight Nordic centres constituting the Nordic consortium for acute type A aortic dissection (NORCAAD) were analysed. Blood group O patients were compared to non-O subjects. The relative frequency of blood groups was assessed with t-distribution, modified for weighted proportions. Multivariable logistic regression was performed to identify independent predictors of 30-day mortality. Cox regression analyses were performed for assessing independent predictors of late mortality. Results. There was no significant difference in the proportions of blood group O between the study populations in the NORCAAD registry and the background population (40.6 (95% CI 37.7-43.4)% vs 39.0 (95% CI 39.0-39.0)%). ABO blood group was not associated with any significant change in risk of 30-day or late mortality, with the exception of blood group A being an independent predictor of late mortality. Prevalence of postoperative complications was similar between the ABO blood groups. Conclusions. In this large cohort of Nordic ATAAD patients, there were no associations between ABO blood group and surgical incidence or outcomes, including postoperative complications and survival.

Impaired von Willebrand factor adhesion and platelet response in thrombospondin-2 knockout mice.

Interactions between collagenous extracellular matrices and von Willebrand factor (VWF) are critical for hemostasis and thrombosis. In the present study, we investigated the contribution of an extracellular matrix (ECM) abnormality to the bleeding diathesis in thrombospondin-2 (TSP2) knockout (KO) mice. First, we performed adoptive bone marrow transplantation and observed that introduction of wild-type (WT) marrow into lethally irradiated TSP2 KO mice did not rescue the bleeding diathesis. However, platelets in transplanted mice displayed an inherent aggregation defect, which complicated interpretation. Second, we performed interposition of arterial segments denuded of endothelium. Denuded TSP2 KO arteries grafted into WT mice remained patent in vivo. In contrast, WT grafts underwent thrombosis and were completely occluded within 24 to 48 hours. The nonthrombogenic property of the TSP2 KO ECM was confirmed in vitro by exposing platelets to TSP2 KO dermal fibroblast (DF)-derived ECM. To further probe the effect of TSP2 deficiency, ECM production and deposition by WT and TSP2 KO DFs was analyzed via polymerase chain reaction, immunofluorescence, and scanning electron microscopy and showed similar patterns. In addition, atomic force microscopy (AFM) analysis of WT and TSP2 KO ECM did not reveal differences in stiffness. In contrast, reduced VWF accumulation on TSP2 KO ECM was observed when matrices were subjected to plasma under physiological flow. AFM utilizing VWF-coated 2-µm beads confirmed the weak binding to TSP2 KO ECM, providing a mechanistic explanation for the lack of thrombus formation. Therefore, our studies show that ECM assembly is critical for interaction of collagen with VWF and subsequent thrombogenic responses.

Deficient Circumferential Growth Is the Primary Determinant of Aortic Obstruction Attributable to Partial Elastin Deficiency.

OBJECTIVE: Williams syndrome is characterized by obstructive aortopathy attributable to heterozygous loss of ELN, the gene encoding elastin. Lesions are thought to result primarily from excessive smooth muscle cell (SMC) proliferation and consequent medial expansion, although an initially smaller caliber and increased stiffness of the aorta may contribute to luminal narrowing. The relative contributions of such abnormalities to the obstructive phenotype had not been defined. APPROACH AND RESULTS: We quantified determinants of luminal stenosis in thoracic aortas of Eln-/- mice incompletely rescued by human ELN. Moderate obstruction was largely because of deficient circumferential growth, most prominently of ascending segments, despite increased axial growth. Medial thickening was evident in these smaller diameter elastin-deficient aortas, with medial area similar to that of larger diameter control aortas. There was no difference in cross-sectional SMC number between mutant and wild-type genotypes at multiple stages of postnatal development. Decreased elastin content was associated with medial fibrosis and reduced aortic distensibility because of increased structural stiffness but preserved material stiffness. Elastin-deficient SMCs exhibited greater contractile-to-proliferative phenotypic modulation in vitro than in vivo. We confirmed increased medial collagen without evidence of increased medial area or SMC number in a small ascending aorta with thickened media of a Williams syndrome subject. CONCLUSIONS: Deficient circumferential growth is the predominant mechanism for moderate obstructive aortic disease resulting from partial elastin deficiency. Our findings suggest that diverse aortic manifestations in Williams syndrome result from graded elastin content, and SMC hyperplasia causing medial expansion requires additional elastin loss superimposed on ELN haploinsufficiency.

mTOR (Mechanistic Target of Rapamycin) Inhibition Decreases Mechanosignaling, Collagen Accumulation, and Stiffening of the Thoracic Aorta in Elastin-Deficient Mice.

OBJECTIVE: Elastin deficiency because of heterozygous loss of an ELN allele in Williams syndrome causes obstructive aortopathy characterized by medial thickening and fibrosis and consequent aortic stiffening. Previous work in Eln-null mice with a severe arterial phenotype showed that inhibition of mTOR (mechanistic target of rapamycin), a key regulator of cell growth, lessened the aortic obstruction but did not prevent early postnatal death. We investigated the effects of mTOR inhibition in Eln-null mice partially rescued by human ELN that manifest a less severe arterial phenotype and survive long term. APPROACH AND RESULTS: Thoracic aortas of neonatal and juvenile mice with graded elastin deficiency exhibited increased signaling through both mTOR complex 1 and 2. Despite lower predicted wall stress, there was increased phosphorylation of focal adhesion kinase, suggestive of greater integrin activation, and increased transforming growth factor-ß-signaling mediators, associated with increased collagen expression. Pharmacological blockade of mTOR by rapalogs did not improve luminal stenosis but reduced mechanosignaling (in delayed fashion after mTOR complex 1 inhibition), medial collagen accumulation, and stiffening of the aorta. Rapalog administration also retarded somatic growth, however, and precipitated neonatal deaths. Complementary, less-toxic strategies to inhibit mTOR via altered growth factor and nutrient responses were not effective. CONCLUSIONS: In addition to previously demonstrated therapeutic benefits of rapalogs decreasing smooth muscle cell proliferation in the absence of elastin, we find that rapalogs also prevent aortic fibrosis and stiffening attributable to partial elastin deficiency. Our findings suggest that mTOR-sensitive perturbation of smooth muscle cell mechanosensing contributes to elastin aortopathy.

Rac2 Modulates Atherosclerotic Calcification by Regulating Macrophage Interleukin-1ß Production.

OBJECTIVE: The calcium composition of atherosclerotic plaque is thought to be associated with increased risk for cardiovascular events, but whether plaque calcium itself is predictive of worsening clinical outcomes remains highly controversial. Inflammation is likely a key mediator of vascular calcification, but immune signaling mechanisms that promote this process are minimally understood. APPROACH AND RESULTS: Here, we identify Rac2 as a major inflammatory regulator of signaling that directs plaque osteogenesis. In experimental atherogenesis, Rac2 prevented progressive calcification through its suppression of Rac1-dependent macrophage interleukin-1ß (IL-1ß) expression, which in turn is a key driver of vascular smooth muscle cell calcium deposition by its ability to promote osteogenic transcriptional programs. Calcified coronary arteries from patients revealed decreased Rac2 expression but increased IL-1ß expression, and high coronary calcium burden in patients with coronary artery disease was associated with significantly increased serum IL-1ß levels. Moreover, we found that elevated IL-1ß was an independent predictor of cardiovascular death in those subjects with high coronary calcium burden. CONCLUSIONS: Overall, these studies identify a novel Rac2-mediated regulation of macrophage IL-1ß expression, which has the potential to serve as a powerful biomarker and therapeutic target for atherosclerosis.